Ipomoeassin-F disrupts multiple aspects of secretory protein biogenesis

AbstractThe Sec61 complex translocates nascent polypeptides into and across the membrane of the endoplasmic reticulum (ER), providing access to the secretory pathway. In this study, we show that Ipomoeassin-F (Ipom-F), a selective inhibitor of protein entry into the ER lumen, blocks the in vitro translocation of certain secretory proteins and ER lumenal folding factors whilst barely affecting others such as albumin. The effects of Ipom-F on protein secretion from HepG2 cells are twofold: reduced ER translocation combined, in some cases, with defective ER lumenal folding. This latter issue is most likely a consequence of Ipom-F preventing the cell from replenishing its ER lumenal chaperones. Ipom-F treatment results in two cellular stress responses: firstly, an upregulation of stress-inducible cytosolic chaperones, Hsp70 and Hsp90; secondly, an atypical unfolded protein response (UPR) linked to the Ipom-F-mediated perturbation of ER function. Hence, although levels of spliced XBP1 and CHOP mRNA and ATF4 protein increase with Ipom-F, the accompanying increase in the levels of ER lumenal BiP and GRP94 seen with tunicamycin are not observed. In short, although Ipom-F reduces the biosynthetic load of newly synthesised secretory proteins entering the ER lumen, its effects on the UPR preclude the cell restoring ER homeostasis.

Download Full-text

Unfolded Protein Response Pathways in Bloodstream-Form Trypanosoma brucei?

Eukaryotic Cell ◽

10.1128/ec.00118-15 ◽

2015 ◽

Vol 14 (11) ◽

pp. 1094-1101 ◽

Cited By ~ 7

Author(s):

Calvin Tiengwe ◽

Abigail E. N. A. Brown ◽

James D. Bangs

Keyword(s):

Er Stress ◽

Unfolded Protein Response ◽

Trypanosoma Brucei ◽

Mammalian Cells ◽

Secretory Pathway ◽

Secretory Protein ◽

Secretory Proteins ◽

African Trypanosomes ◽

Unfolded Protein ◽

Protein Response

ABSTRACT The unfolded protein response (UPR) is a stress mechanism to cope with misfolded proteins in the early secretory pathway, the hallmark being transcriptional upregulation of endoplasmic reticulum (ER) molecular chaperones such as BiP and protein disulfide isomerase. Despite the lack of transcriptional regulation and the absence of the classical UPR machinery, African trypanosomes apparently respond to persistent ER stress by a UPR-like response, including upregulation of BiP, and a related spliced leader silencing (SLS) response whereby SL RNA transcription is shut down. Initially observed by knockdown of the secretory protein translocation machinery, both responses are also induced by chemical agents known to elicit UPR in mammalian cells (H. Goldshmidt, D. Matas, A. Kabi, A. Carmi, R. Hope, S. Michaeli, PLoS Pathog 6:e1000731, 2010, http://dx.doi.org/10.1371/journal.ppat.1000731 ). As these findings were generated primarily in procyclic-stage trypanosomes, we have investigated both responses in pathogenic bloodstream-stage parasites. RNA interference (RNAi) silencing of the core translocon subunit Trypanosoma brucei Sec61α (TbSec61α) failed to induce either response. Interestingly, cell growth halted within 16 h of silencing, but sufficient TbSec61α remained to allow full competence for translocation of nascent secretory proteins for up to 24 h, indicating that replication is finely coupled with the capacity to synthesize and transport secretory cargo. Tunicamycin and thapsigargin at concentrations compatible with short-term (4 h) and long-term (24 h) viability also failed to induce any of the indicators of UPR-like or SLS responses. Dithiothreitol (DTT) was lethal at all concentrations tested. These results indicate that UPR-like and SLS responses to persistent ER stress do not occur in bloodstream-stage trypanosomes.

Download Full-text

Two Distinctly Localized P-Type ATPases Collaborate to Maintain Organelle Homeostasis Required for Glycoprotein Processing and Quality Control

Molecular Biology of the Cell ◽

10.1091/mbc.02-06-0090 ◽

2002 ◽

Vol 13 (11) ◽

pp. 3955-3966 ◽

Cited By ~ 61

Author(s):

Shilpa Vashist ◽

Christian G. Frank ◽

Claude A. Jakob ◽

Davis T.W. Ng

Keyword(s):

Quality Control ◽

Unfolded Protein Response ◽

Secretory Pathway ◽

Ion Homeostasis ◽

Unfolded Protein ◽

The Unfolded Protein Response ◽

Coa Reductase ◽

Protein Response ◽

P Type ◽

Er Homeostasis

Membrane transporter proteins are essential for the maintenance of cellular ion homeostasis. In the secretory pathway, the P-type ATPase family of transporters is found in every compartment and the plasma membrane. Here, we report the identification of COD1/SPF1(control of HMG-CoA reductase degradation/SPF1) through genetic strategies intended to uncover genes involved in protein maturation and endoplasmic reticulum (ER)-associated degradation (ERAD), a quality control pathway that rids misfolded proteins. Cod1p is a putative ER P-type ATPase whose expression is regulated by the unfolded protein response, a stress-inducible pathway used to monitor and maintain ER homeostasis. COD1 mutants activate the unfolded protein response and are defective in a variety of functions apart from ERAD, which further support a homeostatic role.COD1 mutants display phenotypes similar to strains lacking Pmr1p, a Ca2+/Mn2+pump that resides in the medial-Golgi. Because of its localization, the previously reported role of PMR1 in ERAD was somewhat enigmatic. A clue to their respective roles came from observations that the two genes are not generally required for ERAD. We show that the specificity is rooted in a requirement for both genes in protein-linked oligosaccharide trimming, a requisite ER modification in the degradation of some misfolded glycoproteins. Furthermore, Cod1p, like Pmr1p, is also needed for the outer chain modification of carbohydrates in the Golgi apparatus despite its ER localization. In strains deleted of both genes, these activities are nearly abolished. The presence of either protein alone, however, can support partial function for both compartments. Taken together, our results reveal an interdependent relationship between two P-type ATPases to maintain homeostasis of the organelles where they reside.

Download Full-text

The unfolded protein response coordinates the production of endoplasmic reticulum protein and endoplasmic reticulum membrane.

Molecular Biology of the Cell ◽

10.1091/mbc.8.9.1805 ◽

1997 ◽

Vol 8 (9) ◽

pp. 1805-1814 ◽

Cited By ~ 277

Author(s):

J S Cox ◽

R E Chapman ◽

P Walter

Keyword(s):

Endoplasmic Reticulum ◽

Unfolded Protein Response ◽

Secretory Pathway ◽

Lipid Synthesis ◽

Secretory Proteins ◽

Endoplasmic Reticulum Membrane ◽

Yeast Saccharomyces Cerevisiae ◽

Unfolded Protein ◽

The Unfolded Protein Response ◽

Protein Response

The endoplasmic reticulum (ER) is a multifunctional organelle responsible for production of both lumenal and membrane components of secretory pathway compartments. Secretory proteins are folded, processed, and sorted in the ER lumen and lipid synthesis occurs on the ER membrane itself. In the yeast Saccharomyces cerevisiae, synthesis of ER components is highly regulated: the ER-resident proteins by the unfolded protein response and membrane lipid synthesis by the inositol response. We demonstrate that these two responses are intimately linked, forming different branches of the same pathway. Furthermore, we present evidence indicating that this coordinate regulation plays a role in ER biogenesis.

Download Full-text

ER stress causes widespread protein aggregation and prion formation

The Journal of Cell Biology ◽

10.1083/jcb.201612165 ◽

2017 ◽

Vol 216 (8) ◽

pp. 2295-2304 ◽

Cited By ~ 28

Author(s):

Norfadilah Hamdan ◽

Paraskevi Kritsiligkou ◽

Chris M. Grant

Keyword(s):

Protein Aggregation ◽

Er Stress ◽

Secretory Pathway ◽

Cellular Protein ◽

Protein Homeostasis ◽

Er Quality Control ◽

Unfolded Protein ◽

The Unfolded Protein Response ◽

Protein Response ◽

Er Homeostasis

Disturbances in endoplasmic reticulum (ER) homeostasis create a condition termed ER stress. This activates the unfolded protein response (UPR), which alters the expression of many genes involved in ER quality control. We show here that ER stress causes the aggregation of proteins, most of which are not ER or secretory pathway proteins. Proteomic analysis of the aggregated proteins revealed enrichment for intrinsically aggregation-prone proteins rather than proteins which are affected in a stress-specific manner. Aggregation does not arise because of overwhelming proteasome-mediated degradation but because of a general disruption of cellular protein homeostasis. We further show that overexpression of certain chaperones abrogates protein aggregation and protects a UPR mutant against ER stress conditions. The onset of ER stress is known to correlate with various disease processes, and our data indicate that widespread amorphous and amyloid protein aggregation is an unanticipated outcome of such stress.

Download Full-text

70 XBP1 DYSREGULATION BY CRISPR/Cas9-MEDIATED GENE EDITING DURING PORCINE EMBRYO EARLY DEVELOPMENT

Reproduction Fertility and Development ◽

10.1071/rdv29n1ab70 ◽

2017 ◽

Vol 29 (1) ◽

pp. 142

Author(s):

K. Gutierrez ◽

W. G. Glanzner ◽

N. Dicks ◽

R. C. Bohrer ◽

L. G. Currin ◽

...

Keyword(s):

Er Stress ◽

Gene Editing ◽

Early Embryo ◽

Early Embryo Development ◽

Unfolded Protein ◽

Guide Rnas ◽

The Unfolded Protein Response ◽

Protein Response ◽

Er Homeostasis

Early developing embryos are very sensitive to their developmental milieu. For instance, variations in temperature, pH, or culture media composition can trigger endoplasmic reticulum (ER) stress. Endoplasmic reticulum stress has been shown to reduce early embryo development and embryo quality. In response to ER stress, embryos activate coping mechanisms, such as the unfolded protein response, to re-establish ER homeostasis. The X box binding protein (XBP1) is one of the main transducers of the unfolded protein response. Under ER stress, XBP1 mRNA is unconventionally spliced by IRE1α to yield its activated isoform (XBP1s), which allows expression of genes involved in protein folding, transport, and degradation. XBP1s has been detected in oocytes and early stage embryos of different species, including Drosophila, Xenopus, zebrafish, mice, and pigs, suggesting an important role during early embryo development. In this study, we used the CRISPR/Cas9 gene editing technology to investigate the effect of XBP1 dysregulation during development of porcine embryos in vitro. Pig zygotes were produced by intracytoplasmic sperm injection using in vitro-matured oocytes. Treatments consisted of (a) Cas9 mRNA (Cas9) + 1 single guide RNAs targeting XBP1 gene region 1 (sgRNA-1); (b) Cas9 + 1 single guide RNAs targeting XBP1 gene region 2 (sgRNA-2); (c) Cas9 + sgRNA-1 + sgRNA-2; (d) Cas9 alone; and (e) sgRNA-1 + sgRNA-2. After injection, embryos were cultured in vitro for 5 to 7 days to assess development and cell numbers. Experiments were repeated 5 or more times, and data were analysed by ANOVA and means compared using Student’s t-test or Tukey–Kramer Honestly Significant Difference test. Embryo cleavage was similar between the groups (a = 59.8 ± 4.9%, b = 58.8 ± 5.3%, c = 68.86 ± 2.2%, d = 66.4 ± 5.9%, and e = 70.10 ± 1.9%), but development to the blastocyst stage was substantially reduced (P < 0.05) in the groups injected with Cas9 + sgRNAs (a = 18 ± 4.5%, b = 16 ± 1.5%, and c = 5.3 ± 2.8%) compared with controls (d = 33.7 ± 6.2% and e = 31.4 ± 1.2%). Moreover, we observed that only 22.7% of the embryos treated with Cas9 + sgRNA-1 + sgRNA-2 were able to develop beyond 8-cell stage compared with 62.5% in the control group injected with Cas9 alone. These findings suggest that XBP1 activity is required for maintenance of ER homeostasis and development of porcine embryos beyond the main period of embryo genome activation.

Download Full-text

ER homeostasis and autophagy

Essays in Biochemistry ◽

10.1042/ebc20170092 ◽

2017 ◽

Vol 61 (6) ◽

pp. 625-635 ◽

Cited By ~ 69

Author(s):

Matthew Smith ◽

Simon Wilkinson

Keyword(s):

Secretory Proteins ◽

Unfolded Protein ◽

Effector Pathway ◽

Autophagy Pathway ◽

The Unfolded Protein Response ◽

And Function ◽

Protein Response ◽

Er Homeostasis ◽

Effector Mechanisms

The endoplasmic reticulum (ER) is a key site for lipid biosynthesis and folding of nascent transmembrane and secretory proteins. These processes are maintained by careful homeostatic control of the environment within the ER lumen. Signalling sensors within the ER detect perturbations within the lumen (ER stress) and employ downstream signalling cascades that engage effector mechanisms to restore homeostasis. The most studied signalling mechanism that the ER employs is the unfolded protein response (UPR), which is known to increase a number of effector mechanisms, including autophagy. In this chapter, we will discuss the emerging role of autophagy as a UPR effector pathway. We will focus on the recently discovered selective autophagy pathway for ER, ER-phagy, with particular emphasis on the structure and function of known mammalian ER-phagy receptors, namely FAM134B, SEC62, RTN3 and CCPG1. Finally, we conclude with our view of where the future of this field can lead our understanding of the involvement of ER-phagy in ER homeostasis.

Download Full-text

Cysteine cross-linking in native membranes establishes the transmembrane architecture of Ire1

The Journal of Cell Biology ◽

10.1083/jcb.202011078 ◽

2021 ◽

Vol 220 (8) ◽

Author(s):

Kristina Väth ◽

Carsten Mattes ◽

John Reinhard ◽

Roberto Covino ◽

Heike Stumpf ◽

...

Keyword(s):

Lipid Bilayer ◽

Cross Linking ◽

Secretory Proteins ◽

Transmembrane Helices ◽

Unfolded Proteins ◽

Unfolded Protein ◽

The Unfolded Protein Response ◽

Protein Response ◽

Er Homeostasis ◽

Protein Load

The ER is a key organelle of membrane biogenesis and crucial for the folding of both membrane and secretory proteins. Sensors of the unfolded protein response (UPR) monitor the unfolded protein load in the ER and convey effector functions for maintaining ER homeostasis. Aberrant compositions of the ER membrane, referred to as lipid bilayer stress, are equally potent activators of the UPR. How the distinct signals from lipid bilayer stress and unfolded proteins are processed by the conserved UPR transducer Ire1 remains unknown. Here, we have generated a functional, cysteine-less variant of Ire1 and performed systematic cysteine cross-linking experiments in native membranes to establish its transmembrane architecture in signaling-active clusters. We show that the transmembrane helices of two neighboring Ire1 molecules adopt an X-shaped configuration independent of the primary cause for ER stress. This suggests that different forms of stress converge in a common, signaling-active transmembrane architecture of Ire1.

Download Full-text

Systematic cysteine-crosslinking in native membranes establishes the transmembrane architecture in Ire1 clusters

10.1101/772772 ◽

2019 ◽

Author(s):

Kristina Väth ◽

Roberto Covino ◽

John Reinhard ◽

Gerhard Hummer ◽

Robert Ernst

Keyword(s):

Lipid Bilayer ◽

Secretory Proteins ◽

Transmembrane Helices ◽

Unfolded Protein ◽

Proteotoxic Stress ◽

Stress Sensors ◽

Obesity And Diabetes ◽

The Unfolded Protein Response ◽

Protein Response ◽

Er Homeostasis

AbstractThe endoplasmic reticulum (ER) is a key organelle of membrane biogenesis and crucial for the folding of both membrane and secretory proteins. Stress sensors of the unfolded protein response (UPR) monitor the unfolded protein load in the ER and convey effector functions for the maintenance of ER homeostasis. More recently, it became clear that aberrant compositions of the ER membrane, referred to as lipid bilayer stress, are equally potent activators of the UPR with important implications in obesity and diabetes. How the distinct signals from lipid bilayer stress and proteotoxic stress are processed by the highly conserved UPR transducer Ire1 remains unknown. Here, we have generated a functional, cysteine-less variant of Ire1 and performed systematic cysteine crosslinking experiments to establish the transmembrane architecture of signaling-active clusters in native membranes. We show that the transmembrane helices of two neighboring Ire1 molecules adopt an X-shaped configuration and that this configuration is independent of the primary cause for ER stress. Based on these findings, we propose that different forms of stress converge in a single, signaling-active conformation of Ire1.

Download Full-text

Dynamic tracking and identification of tissue-specific secretory proteins in the circulation of live mice

Nature Communications ◽

10.1038/s41467-021-25546-y ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Kwang-eun Kim ◽

Isaac Park ◽

Jeesoo Kim ◽

Myeong-Gyun Kang ◽

Won Gun Choi ◽

...

Keyword(s):

Secretory Pathway ◽

Ex Vivo ◽

Secretory Protein ◽

Spatiotemporal Dynamics ◽

Whole Body ◽

Accurate Information ◽

Secretory Proteins ◽

Specific Cell

AbstractSecretory proteins are an essential component of interorgan communication networks that regulate animal physiology. Current approaches for identifying secretory proteins from specific cell and tissue types are largely limited to in vitro or ex vivo models which often fail to recapitulate in vivo biology. As such, there is mounting interest in developing in vivo analytical tools that can provide accurate information on the origin, identity, and spatiotemporal dynamics of secretory proteins. Here, we describe iSLET (in situ Secretory protein Labeling via ER-anchored TurboID) which selectively labels proteins that transit through the classical secretory pathway via catalytic actions of Sec61b-TurboID, a proximity labeling enzyme anchored in the ER lumen. To validate iSLET in a whole-body system, we express iSLET in the mouse liver and demonstrate efficient labeling of liver secretory proteins which could be tracked and identified within circulating blood plasma. Furthermore, proteomic analysis of the labeled liver secretome enriched from liver iSLET mouse plasma is highly consistent with previous reports of liver secretory protein profiles. Taken together, iSLET is a versatile and powerful tool for studying spatiotemporal dynamics of secretory proteins, a valuable class of biomarkers and therapeutic targets.

Download Full-text

Efficient binding of regulated secretory protein aggregates to membrane phospholipids at acidic pH

Biochemical Journal ◽

10.1042/bj3380289 ◽

1999 ◽

Vol 338 (2) ◽

pp. 289-294 ◽

Cited By ~ 3

Author(s):

Jean LAINÉ ◽

Denis LeBEL

Keyword(s):

Secretory Pathway ◽

Membrane Lipids ◽

Secretory Granules ◽

Ph Dependence ◽

Secretory Protein ◽

Secretory Proteins ◽

Zymogen Granule ◽

Membrane Phospholipids ◽

Aggregation Assay

Some regulated secretory proteins are thought to be targeted to secretory granules through an acidic-dependent aggregation in the trans-Golgi network. In this report we use pancreatic zymogens, a paradigm of regulated proteins, to test this hypothesis, because they qualitatively aggregate upon acidification in vitro. Pig zymogens were found to start to aggregate significantly at pH ∼ 6.0, a pH slightly lower than that at which rat zymogens aggregate, but still compatible with the pH of the cell-sorting compartments. When pig zymogen granule membranes were mixed with the zymogens in the aggregation assay, membranes that normally floated on 1 M sucrose were observed to be pelleted by the aggregating zymogens. Rat membranes were pelleted by pig zymogens and vice versa. Igs, typical constitutively secreted proteins, which needed chemical cross-linking to serve as an aggregated protein control, pelleted membranes almost independently of pH. Corresponding cross-linked zymogen-binding ability and pH dependence was unaffected by the chemical modification. Membranes treated with sodium carbonate, pH 11, or with protease K, were still pelleted by zymogens, suggesting that the aggregated zymogens bound to membrane lipids. This hypothesis was confirmed by the efficient pelleting of unilamellar vesicles composed of granule membrane lipids. Vesicles composed of single classes of phospholipids were also pelleted, but with various efficacies. We conclude that pancreatic zymogen aggregates, formed under the acidic conditions of the secretory pathway sorting compartments, have the capacity to bind firmly to membranes through their phospholipid constituents.

Download Full-text